Nucleocytoplasmic transport through the nuclear pore complex (NPC) is central to each step in eukaryotic gene expression. During transcription and RNA processing, an mRNA ribonucleoprotein particle (mRNP) is formed for export, translation and cytoplasmic turnover. Our research focuses on determining the molecular sequence of events required for mRNA export through NPCs, and for coordinating export with cytoplasmic fates to regulate gene expression. We hypothesize that mRNP metabolism is directed by its specific protein complement and that remodeling of that composition is necessary at multiple stages along the pathway. We have used a powerful combination of S. cerevisiae and mammalian cell culture model systems to study the highly conserved machinery involved in mRNA export. We defined key players in generating export-competent mRNPs and pinpointed roles of phenylalanine-glycine repeats in NPC translocation. We identified a mechanism for mRNP remodeling at the cytoplasmic NPC face through activation of the DEAD-box protein Dbp5 by Gle1 and inositol hexakisphosphate. We discovered that Glel is a multi-functional regulator of Dbps, linking mRNA export and translation. Most recently, we uncovered the molecular defects in NPC and Gle1 function that are causal to lethal congenital contracture syndrome-1. We are now uniquely positioned to further investigate novel aspects of NPC structure/function in connection to the full gene expresion cycle, and give new insight into human disease. Using a multidisciplinary approach, the molecular determinants for mRNP remodeling at the NPC will be dissected, including the novel requirement for Gle1 self association. We will establish a robust system with a stress-induced model transcript to elucidate for the first time how mRNP composition directs fate. Finally, we will employ yeast, mammalian cell culture and zebrafish models to identify the nucleocytoplasmic transport perturbations in lethal arthrogyposis with anterior horn cell disease, amyotrophic latheral sclerosis, atrial fibrillation and aging - each connected to NPC and/or Gle1 dysfunction. Together, these studies will define the machinery controlling critical steps in the mRNA life cycle, and impact paradigms for altered mRNA transport and metabolism in human disease. RELEVANCE (See instructions): Understanding how proteins and RNA move between the nucleus and cytoplasm will provide novel insights into the pathology of multiple human diseases. Altered expression of nuclear transport factors is implicated in cancers, viral infections, developmental defects, heart and immunological diseases; Gle1 is causally linked to developmental organogenesis and progressive neurodegenerative diseases. These studies will provide critical knowledge for the ultimate identification and analysis of therapeutic strategies and targets.